It is a well established fact that the presence of essential minerals plays an important role in animal feeding. The availability and assimilation of these feed components is reflected in the performance of the animal and the excretion in the manure. Due to the increase of the intensive livestock production, manure production causes environmental problems, partly because of its phosphates. Moreover, legislation concerning the manure problematic, especially the phosphorus content of the manure, entails expenses, which makes it necessary to reduce phosphorus excretion in the environment.
Phosphorus is added to the feed by different plant raw materials, animal byproducts and inorganic phosphorus.
Phytic acid or phytate is the hexa-phosphorus ester of inositol (myo-inositol hexakisphosphate), found in many seeds and cereals. It acts as the primary storage form of both phosphorus and inositol and accounts for more than 50% of the total phosphorus content (LOLAS et al., 1976; SAUVEUR, 1989). Oilseeds can contain up to 5.2% of phytin (REDDY et al., 1982).
However, phytin phosphorus of plant raw materials is poorly digested by monogastric animals such as poultry, swine and man because of their simple intestinal tract: they lack or have only low intestinal phytase activity to catalyze the hydrolysis of these phytates in their intestine and phytin phosphorus released in the colon is excreted in the environment. Plant raw materials are by these means poor phosphorus sources for animal feeding, and additional phosphorus has to be included in the diet by animal byproducts or inorganic phosphates.
Moreover, phytin is considered as an anti-nutritional factor due to its chelating properties: it binds many multivalent cations such as Ca.sup.2+, Fe.sup.3+, Mg.sup.2+ and Zn.sup.2+ by forming insoluble complexes therewith and hence reduces the bioavailability and absorption of these essential dietary minerals. Besides, complexation of proteins with phytin (COSGROVE, 1966) obstructs enzymic protein digestion.
The negative effects of phytin on phosphorus and mineral metabolism, coupled to a high phosphorus excretion in the environment and the existence of a legislation concerning phosphorus excretion, makes it necessary to render phytin phosphorus bioavailable.
Phytin or phytates can be hydrolysed enzymatically by phytases either present in plant raw materials or produced by micro-organisms.
The enzyme phytase (myo-inositol hexaphosphate phosphohydrolase E.C. 3.1.3.8.) hydrolyses, under proper conditions, phytic acid or phytate into inorganic phosphate, inositol and inositol mono- to penta-phosphates.
Phytase is widely distributed in plants and microorganisms, especially fungi, but is found only in insignificant quantities in the intestinal tract of monogastric animals.
Plant phytases are, due to their low pH-stability and narrow pH-activity range (SUTARDI & BUCKLE, 1986; LOLAS & MARKAKIS, 1977) rapidly inactivated in the digestive tract of monogastric animals, and their in vivo efficiency is low (EECKHOUT & DE PAEPE, 1991). They are therefore of minor importance for the animal compound feed formulation.
On the contrary, some microbial phytases have a broad pH-stability and pH-activity range, by which phytin can be hydrolysed more efficiently in the intestinal tract of the animal. For this reason, microbial phytase production processes have been developed to upgrade phytin phosphorus in animal diets by application of phytase that can tolerate the acid conditions in the stomach. Nevertheless, thermostability of phytase is still too low to withstand the high temperatures (70.degree.-80.degree. C.) achieved during the compound feed manufacturing process. In view thereof, it may be necessary to apply an overdosis of 30%.
It has already been demonstrated in vivo that the addition of fungal phytase can improve the assimilation of phytin phosphorus and minerals, by which the phosphorus conversion coefficient is increased and the phosphorus amount in feeds and manure is reduced.
Microbial phytase activity is well documented. Next to bacterial (GREAVES et al., 1967; IRVING & COSGROVE, 1971; POWAR & JAGANNATHAN, 1982) and yeast phytases (NAYINI & MARKAKIS, 1984), phytases are mainly found in molds, in particular Aspergillus strains (SHIEH & WARE, 1968; YAMAMOTO et al., 1972; YOUSSEF et al., 1987). Most of these strains, and other microorganisms, also produce acid phosphatases. Although some phosphatases have been called phytases, they are rather nonspecific and their hydrolytic activity to phytin is low compared to other organic phosphates.
Dependent on fermentation conditions, Aspergillus ficuum NRRL 3135 wild type strain produces a mixture of extracellular phosphatases and phytase(s). Phytase and acid phosphatase synthesis can be regulated by the phosphorus concentration, according to methods known in the art (SHIEH et al., 1969; ULLAH and CUMMINS, 1987). Recently published patent applications claim the use of genetic engineered Aspergillus ficuum NRRL 3135 and Aspergillus niger strains to obtain a high production level of phytase (E.P. 0 420 358 A1). Cloning of acid phosphatase has also been mentioned in this document.
Purification of crude A. ficuum NRRL 3135 phytase culture broth (ULLAH & GIBSON, 1987) gives a phytase with two distinct pH-optima: highest activity is found at pH 5.0-5.5 while the second activity peak (60% of pH 5.0 activity) occurs at pH 2.2.
ULLAH & CUMMINS (1987) purified an Aspergillus ficuum NRRL 3135 acid phosphatase (orthophosphoric monoester phosphohydrolase E.C. 3.1.3.2.) with a pH-optimum of 2.5. The acid phosphatase is 65% less active at pH 4.5 and is virtually inactive at pH 6.0. Another acid phosphatase was purified by ULLAH & CUMMINS (1988) with a pH-optimum of 6.0.
Both acid phosphatases were unable to accommodate phytate as a substrate although they exhibit a broad substrate selectivity on various organic phosphomonoesters (ULLAH & CUMMINS, 1988). IRVING & COSGROVE (1974) on the contrary mentioned a side activity (16%) of A. ficuum acid phosphatase with pH-optimum 2.2 on phytate.
Recent publications of ZYLA (1993) describe the action of acid phosphatase in the presence of phytase from Aspergillus niger on feed raw materials and feed at different pH values.
A. ficuum NRRL 3135 phytase and acid phosphatases not only differ from each other in substrate specificity and pH-optima, but also in temperature optima. Phytase develops highest activity at 58.degree. C. and looses all activity at 68.degree. C. The acid phosphatase with pH-optimum 2.5 has a temperature optimum of 63.degree. C. and still retains 88% of its catalytic acitity at 70.degree. C.
The temperature optimum of the acid phosphatase with pH-optimum 6.0 is also 63.degree. C., but the enzyme looses 92% of its activity at 70.degree. C. From this, it can be concluded that the acid phosphatase with pH-optimum 2.5 is active at a higher temperature range than the phytase and the acid phosphatase with pH-optimum 6.0.
A non-purified intracellular acid phosphatase from a waste mycelium of Aspergillus niger has a pH-optimum of from 1.8 to 2.6 and a temperature optimum of 60.degree. C. Residual acid phosphatase acitivity at pH 4.5 was still 85% of its maximum (ZYLA et al., 1989).
Aspergillus ficuum phytase activity is supplemented to pig and broiler feed according to the desired phytin and faecal phosphorus reduction, combined with a good performance of the animal including growth and feed conversion. A phytase activity of 500 to 1000 Units (pH 5)/kg feed is mentioned for pig and broiler diets (SIMONS et al., 1990), in which an activity of 500 Units/kg is equal to 0.8 g phosphorus/kg in pig feed and 1.0 g phosphorus/kg in chicken feed (BORGGREVE, 1991; VAHL, 1991). Recommended addition of phytase to pig feed is limited to 600 Units/kg because of a decreasing effect of additional units (Natuphos manual, Gist-Brocades).
In vitro hydrolysis of phytin in the plant raw materials wheat bran and soya beans with non-purified intracellular Aspergillus niger acid phosphatase is completed after 2 h at a respective dosage of 12000 and 30000 Units/kg, 40.degree. C. and pH 4.5, while phytin hydrolysis in broiler feed is completed after 4 h reaction time under the same conditions. This reflects the inefficiency of the acid phosphatase to phytin degradation (ZYLA et al., 1989).
ZYLA & KORELESKI (1993) indicated that the in vitro action of acid phosphatase additional to phytase of Aspergillus niger on rapeseed and soya bean is influenced by the pH of incubation. The incubations were performed at a relatively high acid phosphatase/phytase ratio (3.5/1 to 62/1 expressed in phytate hydrolyzing activity).
ZYLA (1993) indicated that the dephosphorylation action of Aspergillus niger acid phosphatase on phytin is different from the action of Aspergillus niger phytase, resulting in an additive action between both enzymes (and a shorter degradation time). The total phosphorus liberation from phytate was indicated to be slower with the more purified phytase preparation (i.e. lower acid phosphatase/phytase ratio). Nevertheless, the most purified preparation still contained a high acid phosphatase/phytase ratio (3.5/1).